TWI435727B - Use of modulating secretion of cytokines - Google Patents

Description

Use to regulate the secretion of cytokines

The present invention relates to a pharmaceutical composition for treating arthritis and regulating cytokine secretion, and more particularly to a pharmaceutical composition for treating arthritis and regulating cytokine secretion by using mangosteen extract as an active ingredient.

Due to the advancement and popularization of medical technology, the global population structure tends to be aging, and public health issues related to chronic diseases in the elderly are derived. Arthritis has long been a chronic disease with high cost of aging society, which can be derived from factors such as aging, obesity or immune diseases. Traditional treatments use steroidal drugs to reduce inflammatory symptoms and suppress the immune system, or use non-steroidal drugs (NSAIDS) such as COX-1 inhibitors, COX-2 inhibitors, celebrex, or ibuprofen ( Ibuprofen) reduces pain and inflammation, but it can cause side effects of varying degrees of severity. A new generation of drugs for the treatment of arthritis, such as disease-modifying anti-rheumatic drugs (DMARDs), such as aminomethyxate, can slow or stop the immune system from attacking joints, or biologics such as etanercept. Because of infliximab, etc., it can block specific molecular pathways in the immune system involved in the inflammatory process, and thus progress slowly. However, the use of these drugs must still pay special attention to the accompanying side effects.

The treatment of arthritis has also gradually turned to natural remedy, and herbal treatment is an important part. Related research shows that triptolide (Tripterygium wilfordii Hook F) (US Patent 5,580,562) and Bupleurum (Bupleurum) (Taiwan patent application TW 97151783) has the effect of treating arthritis. However, there is still a need to develop novel drugs with good therapeutic effects and few side effects.

Mangosteen ( Garcinia mangostana L.) is a plant specializing in Southeast Asia. Local traditional medicine has been used for the treatment of skin infections and wound treatment for many years. Matsumoto et al. purified α-mangostin, β-mangostin, γ-mangostin, and methyl-β-mangostin from mangosteen peel, and studied the inhibitory effect of this compound on various stages of the cell cycle. The compound has anti-cell proliferation effect and anti-tumor effect (Matsumoto K., et al., Xanthones induce cell-cycle arrest and apoptosis in human colon cancer DLD-1 cells, Bioorg. Med. Chem. 2005, 13, 6064-6069 .).

Mangosteen has also been studied in breast cancer (Moongkarndi P., et al, Antiproliferation, antioxidation and induction of apoptosis by Garcinia mangostana (mangosteen) on SKBR3 human breast cancer cell line, J. Ethnopharmacol. 2004, 90(1): 161-6) , anti-allergic drugs (Nakatani K., et al., Inhibitions of histamine release and prostaglandin E2 synthesis by mangosteen, a Thai medicinal plant, Bio Pharm Bull , 2002 (9): 1137-41.), and muscle-related diseases (WO In the field of 2007/128465), it is also developed as a nutritional supplement for daily life (WO 2006/060578) and cosmetics (WO 2007/002666).

In one aspect of the invention, the invention provides a pharmaceutical composition for treating arthritis, comprising an extract of Mangosteen ( Garcinia mangostana L.) as an active ingredient.

In another aspect of the invention, the present invention provides a pharmaceutical composition for treating arthritis, comprising a compound of the following formula (I) or a salt or ester thereof as an active ingredient, Wherein R ₁ and R ₂ may be the same or different and are each H or a C ₁ -C ₆ alkyl group.

The present invention further provides a use of a mangosteen extract for the manufacture of a medicament for the treatment of arthritis, and a use of a compound of the formula (I) for the manufacture of a medicament for the treatment of arthritis.

In one aspect of the invention, the invention provides a pharmaceutical composition for modulating cytokine secretion, comprising an extract of Mangosteen ( Garcinia mangostana L.) as an active ingredient.

In another aspect of the invention, the present invention provides a pharmaceutical composition for regulating secretion of a cytokine, comprising a compound of the formula (I) or a salt or ester thereof as an active ingredient,

The present invention further provides a use of a mangosteen extract for the manufacture of a medicament for regulating secretion of a cytokine, and a use of a compound of the formula (I) for the manufacture of a medicament for regulating secretion of a cytokine.

The specific embodiments of the present invention are described in detail below, but the following embodiments are only used to further disclose the technical content of the present invention, and should not limit the scope of the invention.

The mangosteen extract of the present invention is extracted from the mangosteen plant body, and the part of the mangosteen peel is preferably used. In an embodiment of the invention, the dried mangosteen peel is selected and extracted with an organic solution. The aforementioned organic solution is not particularly limited and may be a C ₁ -C ₁₂ alcohol such as methanol, ethanol, propanol, isopropanol, butanol, 2-butanol, pentanol, hexanol, heptanol, octanol, anthracene. Alcohol, decyl alcohol, undecyl alcohol or dodecyl alcohol, etc.; or aromatic hydrocarbons such as benzene, toluene or xylene. In one embodiment of the invention, an aqueous ethanol solution is used for extraction. The concentration of the aqueous ethanol solution may be from 10 to 90%, preferably from 20 to 80%, more preferably from 50 to 75%. In one embodiment of the invention, extraction is carried out using a heated aqueous solution of ethanol at a temperature of about 80 to 85 °C. In another embodiment, prior to the extraction of the organic solution solution, the dried mangosteen may be first treated with an equal weight of hot water having a temperature of about 90 ° C or higher, preferably between 95 ° C and 100 ° C. The hot water treatment before the extraction of the organic solution can remove impurities such as color, viscosity, moisture absorption, etc. in the mangosteen. After the above extract is dried, a crude crystal is obtained which contains various mangostin. The above extraction method can be carried out once to obtain a crude extract or crude crystal, or repeated several times to obtain a relatively pure mangosteen extract or crystal.

The mangosteen extract obtained by the above extraction method is subjected to high performance liquid chromatography (HPLC) analysis by means of silica chromatography to obtain a compound of the formula (I) shown below.

Wherein R ₁ and R ₂ may be the same or different and are each H or a C ₁ -C ₆ alkyl group.

In the compound of the above formula (I), when R ₁ is a methyl group and R ₂ is hydrogen, it is α-mangostin; when both R ₁ and R ₂ are a methyl group, it is β-mancelin ( Mangostin); and when both R ₁ and R ₂ are hydrogen, it is γ-mangostin.

The compound of the above formula (I) may be a salt or an ester, and is not particularly limited as long as it does not affect the therapeutic effect of the compound of the formula (I) for treating arthritis and regulating cytokine secretion, such as sodium salt, potassium salt, a salt of a lithium salt, a magnesium salt, a calcium salt, an ammonium salt, a carbonate, a nitrate, a hydrogencarbonate, a hydrochloride, a sulfate, a phosphate, or a citrate; or, for example, a methyl ester, an ethyl ester, or a C Esters of esters, butyl esters, amyl esters, methyl acetate, ethyl acetate, butyl formate, butyl acetate, butyl valerate, butyl propionate, methyl butyrate, ethyl butyrate and the like.

The pharmaceutical composition of the present invention may comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient or other additive, depending on the mode of administration and according to the practice in the art. The route of administration may include oral administration, transdermal administration, intraperitoneal administration, intravenous administration, nasal administration, or ocular administration, and is preferably oral. The dosage can be determined by the relevant medical personnel according to the common knowledge in the technical field according to the patient's age, weight, health status, disease type, disease progression, and affected parts. The pharmaceutical composition of the present invention can also be administered alone or in combination with other agents, and the administration course can be carried out according to a pharmacy routine.

The arthritis of the present invention means a condition in which the joint is inflamed, however, the symptoms of inflammation may also occur in tendons, ligaments, hard bones, cartilage, or muscle tissue. The arthritis described may be caused by an external environment, such as infectious arthritis caused by a viral or bacterial infection, or may be caused by abnormalities in the immune system or genetic variations, such as rheumatoid arthritis, childhood. Juvenile idiopathic arthritis, systemic lupus erythematosus, and the like. The arthritis described also includes osteoarthritis, fibromyalgia, scleroderma, spondyloarthropathies, gout, rheumatic polymyalgia ( Polymyalgia rheumatica), polymyositis, psoriatic arthritis, bursitis, or tendonitis. However, the arthritis of the present invention is not limited to the diseases described herein, and inflammation of joints or related tissues caused by any cause is included in the arthritis of the present invention.

Factors that cause arthritis, many studies have shown that it is related to cytokines, such as tumor necrosis factor α (TNF-α) and interleukins (IL), such as IL-1, IL-4, IL-6 et al. (Tracey D, et al., Tumor necrosis factor antagonist mechanisms of action: a comprehensive review, Pharmacology & Therapeutics 117 (2008) 244-279.). Tracey recalls that several arthritis studies have indicated that the receptor effector of nuclear factor κ-B ligand (RANKL) causes osteoclast effect by TNF, IL-1, IL-6, It is promoted by IL-17 and other cytokines and is inhibited by interferon IFNγ and interleukin IL-4. The increase in the ratio of RANKL to its receptor in rheumatoid arthritis is thought to be caused by increased osteoclast activity. (Tracey D, et al., Tumor necrosis factor antagonist mechanisms of action: a comprehensive review, Pharmacology & Therapeutics 117 (2008) 244-279.). Therefore, for a drug candidate for treating arthritis, the effect of treating arthritis can be evaluated by detecting whether the drug candidate causes a decrease in the content or secretion amount of cytokines TNF-α, IL-4, IL-6 in the living body.

A number of other studies have established animal models of arthritis, such as Thorbecke et al. using collagen-induced arthritis (CIA) animal models, showing that inhibition of TNF significantly slows the progression of arthritis (Thorbecke GJ, et al Involvement of endogenous tumor necrosis factor alpha and transforming growth factor beta during induction of collagen type II arthritis in mice. Proc Natl Acad Sci USA 89, (1992) 7375-7379.); or Mazzon et al. The carrageennan-induced paw edema model effectively evaluates the role of TNF-α during inflammation (Mazzon E., et al., Effect of tumour necrosis factor-αreceptor 1 genetic deletion on carrageenan-induced acute inflammation:a Comparison with etanercept, British Society for Immunology, Clinical and Experimental Immunology, 153 (2008): 136-149.). The entire disclosure of this document is incorporated by reference in its entirety.

The present inventors conducted the following examples according to the arthritis animal model and the arthritis cell strain established in the prior literature, and evaluated the mangosteen extract of the present invention and the compound of the formula (I) or a salt or ester thereof isolated from the mangosteen extract as a treatment. The potential of active ingredients in arthritis. In the following cell tests and arthritic animal models, the mangosteen extract of the present invention and the compound of formula (I) or a salt or ester thereof isolated from the extract significantly occludes the cytokines TNF-α, IL-4, IL- The content of 6 is reduced and the arthritis symptoms of the animal model are slowed down, and the arthritis effect is well treated, especially α-mangostin, β-mangostin and γ-mangostin. Moreover, because mangosteen extract is a product refined from agricultural waste, it has the advantages of being cheap, safe, and can be taken orally, and has advantages in medical applications.

[Example 1] Method for producing mangosteen extract

(a) Mangosteen extract:

Mangosteen peel dry matter 60 kg is added to water 600 liters, heated to above 90 ° C, and stirred for one hour to wash, remove color, viscosity, moisture absorption and other impurities in the heating and washing process. The washed residue of the peel was added to a 600 liter 50% ethanol solution, heated to 80-85 ° C and stirred for one hour and then filtered. The filtrate was allowed to stand at room temperature overnight to precipitate a crude crystal. Filtration gave crude crystals. The separated crude crystals were washed with 10 liters of 50% ethanol, and vacuum dried at 60 ° C to obtain 2.4 kg of crude mangosteen crystal powder.

(b) Separation of mangosteen:

2.4 kg of the crystal powder was added to 24 liters of ethanol, and stirred at room temperature for one hour to dissolve. The solution was slowly added dropwise to 36 liters of water while stirring at room temperature, and stirring was continued at 80 to 85 ° C for one hour, and then continued. Stir and slowly cool to room temperature. Continue to stir at room temperature overnight to crystallize the mangosteen, and crystallize it and wash it with 50% aqueous solution of ethanol, and dry it in vacuo to obtain a composition of mangostin (containing 75-85% of α-mangosin, γ-mangosteen 7 to 15%).

[Example 2] Preparation of in vitro cell line

U937 cell line

A human myeloid leukemia cell line U937 (Rockville, MD) from the American Type Culture Collection (ATCC) was used. The cell strain was cultured in RPMT 1640 medium containing 5% CO ₂ and 10% fetal calf serum (FCS) at 37 ° C, maintained in an exponential growth state. For differentiation induction, the cell strain was cultured in a T150 flask containing 50 ng/ml PMA (Sigma) at an initial concentration of ⁴ x 10 ⁵ cells/ml for 24 hours. It was then transferred to the same medium without PMA and cultured for an additional 48 hours. The flask was then lightly scraped off with a rubber policeman (Bellco Glass, Vineland, NJ) and cells were collected for use in the following examples.

EL-4 cell line

A mouse T lymphoma cell line EL-4 (Rockville, MD) from the American Type Culture Collection (ATCC) was used. This cell strain was cultured in RPMT 1640 medium containing 5% CO ₂ and 10% fetal bovine serum (FCS) at 37 ° C, maintained in an exponential growth state, and cells were collected for use in the following examples.

[Example 3] Inhibition of IL-4 by Mangosteen Extract and Analysis of Cytotoxicity

IL-4 inhibition

In a 96-well plate, 1×10 ⁴ EL-4 cells were implanted, and 30 μl of the mangosteen extract obtained in Example 1 at a concentration of 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, and 10 μg/ml, respectively, at 37 The reaction was carried out at ° C for 2 hours, and then 20 μl of PMA (1.5 ng/well) and calcimycin (A23187, Sigma) (15 ng/well) were added to each well, and cultured at 37 ° C under 5% CO ₂ . The cells were 18 hours. On the next day, the cell culture medium was collected, and its IL-4 content was measured by ELISA (IL-4 duoset, R&D, Minneapolis, MN). The results are shown in Table 1 and Figure 1. Based on the control group containing PMA (phorbol 12-myristate 13-acetate, Sigma) and calcimycin solution alone, the IL-4 secretion was expressed as a percentage. ratio.

Cytotoxicity analysis

The IL-4 inhibitory test of the above example was taken out and the remaining cells were taken out, and 1 mg/ml of MTT (3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltrazolium, Sigma was added at 37 °C. The crystals obtained were added for 1 hour, and 100 μl of DMSO was added to dissolve the crystals. The absorbance (OD ₅₆₀ ) of the dissolved crystals was measured by ELISA Reader (Tecan Spectrafluor plus, Swizerland). The results are shown in Table 1 and Figure 1, and the cell viability was shown as a percentage based on a control group containing PMA alone and a calcimycin solution.

[Example 4] Inhibition of TNF-α by Mangosteen Extract and Analysis of Cytotoxicity

TNF-α inhibition

In a 96-well plate, 1.6× ^{10 5} U937 cells differentiated as in Example 2 were equally dispensed in each well. Further, according to the formulation shown in Table 2 below, the mangosteen extract obtained in Example 1 was mixed with the above cells, and cultured at 37 ° C, 5% CO ₂ for 30 minutes, and then 200 ng / ml of fat was added to each medium. A lipopolysaccharide (LPS) (in phosphate buffered saline (PBS)) was further cultured for 4 hours. The medium was collected by an analytical kit (R&D system, Minneapolis, MN) for analysis of TNF-. The results were calculated by GraFit analysis as shown in Table 2 and Figure 2. The values in Table 2 are based on a control group containing LPS solution alone (0.1 μg/ml), and the percentages show inhibition of TNF-α at various concentrations. The IC ₅₀ was obtained at 4.8 μg/ml.

Cytotoxicity analysis

The procedure of Example 3 was followed, but according to the formulation of Table 2, the results are shown in Table 2 and Figure 2. Similarly, the values in Table 2 are based on a control group containing LPS solution alone (0.1 μg/ml), and the percentages show cell viability at each concentration.

[Example 5] TNF-α inhibition and cytotoxicity of α,β,γ-mangostin

The differentiated U973 cell strain obtained in Example 2 was used, and the α-mancoside and β-mancoside at concentrations of 3.1, 6.3, 12.5, 25.0 μg/ml were tested as in Example 4, and the concentrations were 7.5, 15.0. TNF-α inhibition of γ-mangostin at 30.0 and 60.0 μg/ml. As a result, as shown in Fig. 3a-3c, the IC _{50 of} α,β,γ-mangostin inhibiting TNF-α was 5.5 μg/ml, 8.3 μg/ml, and 4.8 μg/ml, respectively.

[Example 6] Inhibition of mannan extract and α-mangosin on TNF-α and IL-6 in mouse plasma induced by LPS

BALA/c mice obtained from the National Laboratory Animal Center (Taiwan) were used. The mice were housed at room temperature 23 ± 2 ° C, turned on for 12 hours, turned off for 12 hours, and 40-70% air humidity. The experimental animals were free to feed solid food and tap water. All experimental animal codes and their breeding are based on the provisions of the Laboratory Animal Management Subcommittee of the Industrial Technology Research Institute (IACUC, ITRI) and the Council of Agriculture (Taiwan).

The above male BALA/c mice were administered intraperitoneally with 300 mg/kg of mangosteen extract of Example 1 and 100 mg/kg of α-mangostin for 30 minutes, respectively. Thereafter, each mouse was intraperitoneally injected with 1 mg/kg LPS and Cremophore EL (BASF) (hereinafter referred to as CrEL) (5% ethanol in 30% CrEL). At 1.5 hours after LPS injection, mouse blood was collected from heparinated eppendrof. The secretion of TNF-α and IL-6 in the plasma of the mice (mouse TNF-αduoset and IL-4 duoset, R&D, Minneapolis, MN) were measured, and the results are shown in Fig. 4a-4b.

[Example 7] Animal model of mangosteen extract in carrageenan-induced paw edema of rats

Long-Evan rats were used from the National Laboratory Animal Center (Taiwan). The animals were housed at room temperature 23 ± 2 ° C, turned on for 12 hours, turned off for 12 hours, and 40-70% air humidity. The experimental animals were free to feed solid food and tap water. All experimental animal codes and their breeding are based on the provisions of the Experimental Animal Management Subcommittee Training of the Industrial Technology Research Institute (IACUC, ITRI) and the Council of Agriculture (Taiwan).

Five of the above-mentioned Long-Evan rats were fasted for 18-20 hours before the experiment, but they were free to drink water. First, the test group was orally administered with Mangosteen extract 40 mg/kg; the control group was administered with 1% CMC (Carboxymethyl Cellulose) carrier; 50mg/kg. After 1 hour, the left hind paw of all rats was injected with carrageenan-physiological saline solution. Within 5 hours of injection of carrageenan, one rat was sacrificed per hour, and the swelling volume of the hind paw of the mouse was measured by a plethysmometer (PV-01, DR instrument, Taiwan).

The inhibition rate of the inflammatory reaction of carrageenan is calculated according to the following formula:

Inhibition rate (%) = (Nt - Nv) / Nv x 100

Nt: the net change in the palm-shaped swelling volume of the hind paw of the test group;

Nv: Net change in the volume of the hind paw of the vehicle control group.

As a result, as shown in Fig. 5, after 3, 4, and 5 hours of injection of carrageenan, the inhibition rates of the 40 mg/kg mangosteen extract group were 24%, 29%, and 29%, respectively. A negative percentage shows the inhibitory effect of the test group. Results data are expressed as mean ± S.E., P < 0.05.

[Example 8] Mangosteen extract in collagen (Collagen)-induced arthritis animal model

Lewis rats were obtained from the National Laboratory Animal Center (Taiwan). The animals were housed at room temperature 23 ± 2 ° C, turned on for 12 hours, turned off for 12 hours, and 40-70% air humidity. The experimental animals were free to feed solid food and tap water. All experimental animal codes and their breeding are based on the provisions of the Experimental Animal Management Subcommittee Training of the Industrial Technology Research Institute (IACUC, ITRI) and the Council of Agriculture (Taiwan).

The tail of the Lewis rats was subcutaneously injected with 50 μg of fetal bovine type II collagen (Collagen) (CII, Chronderx) in a complete Freund's adjuvant (CFA, Sigma). On the same day, the ankle joint of the rat was marked as the basis for the measurement of the volume of the foot. Seven days after the primary immunization described above, all rats were reinjected with 100 μg of CII in incomplete Freund's adjuvant (IFA, Sigma). The above-mentioned rats were divided into three groups, and 100 mg/kg of mangosteen extract and 30 mg/kg were orally administered daily on the day of induction of the above-mentioned arthritis. And 1% CMC (Carboxymethyl Cellulose) solution (carrier) until the end of the experiment. After the above-mentioned reinjection of CII, the onset of arthritis in the rats was investigated daily, characterized by erythema and/or swelling of the soles of the feet. The incidence and severity of arthritis were measured 3 times a week and assessed by the arthritis grading system. Foot volume and body weight were measured twice a week throughout the experiment. According to the swelling of the tissues around the joints and the degree of erythema, the clinical arthritis characteristics of each foot can be scored as 0-4 scores: 0 means that the soles of the feet are not swollen and no erythema is present; 1 means that the feet have slight erythema, or toes, fingers One joint of the ankle or wrist joint is swollen; 2 indicates erythema on the sole of the foot, and more than 2 joints on the toe or finger are swollen, or moderate erythema and swelling in the ankle or wrist joint; 3 indicates swelling of the sole of the foot, limiting The use of the ankle joint, the foot can not step; 4 indicates excessive swelling of the sole of the foot, and the ankle joint is stiff.

16 is the maximum score in the combined arthritis score for each animal (Ref. Rosloniec, EF, Cremer, M, Kang, A and Myers, LK. Collagen-Induced Arthritis. Current protocols in Immunology (1996) 15.5.1-15.5 .twenty four.).

The above-mentioned foot volume is expressed as a volume change plethysmometer in terms of the average volume of each rat. A precision balancer (METTLLER TOLDEO, PB1501) weighing 0.1 g was measured.

The result is shown in Figure 6. Data are presented as mean ± SEM, and differences between groups were analyzed by Student's t-test compared to vehicle treated groups. P < 0.05 was considered to be statistically significant.

The results from Fig. 6 show that the first visible arthritis characteristic on the 9th day after immunization, the carrier treatment group reached the maximum arthritis score and swelling of the paw on the 19th day, and then gradually subsided. The treatment group of the mangosteen extract was treated with the carrier treatment group for 1 day. The overall incidence of the vehicle treatment group was on the 12th day, and the treatment group of the mangosteen extract reached only 67% on the 14th day until the end of the experiment. Celebrex The incidence of the treatment group from day 14 was 86% until the end of the experiment.

It is known from the experimental results that the treatment group of mangosteen extract reduces the incidence of collagen-induced arthritis patterns. The average score of arthritis severity of the carrier treatment group was 6.88, and the treatment group of mangosteen extract was reduced to 3.50, Celebrex. The processing group is 3.0. The change in the volume of the foot shows a similar trend to the score.

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Figure 1 shows the effect of the mangosteen extract on inhibiting IL-4 secretion and cytotoxicity in vitro .

Figure 2 shows the effect of cynomolgus extract on the inhibition of TNF-α secretion and cytotoxicity in vitro .

Figure 3a shows the effect of α-mangostin inhibiting TNF-α secretion and cytotoxicity in vitro ;

Figure 3b shows the effect of β-mangostin inhibiting TNF-α secretion and cytotoxic mangostin in vitro ;

Figure 3c shows that the effect of mangostin (-mangostin) inhibiting TNF-α secretion and cytotoxicity in vitro .

Figure 4a shows the effect of mangosteen extract and α-mangosin inhibiting TNF-α in lipopolysaccharide (LPS)-induced mouse plasma;

Figure 4b shows the effect of mangosteen extract and α-mangosin inhibiting IL-6 secretion in lipopolysaccharide (LPS)-induced mouse plasma.

Figure 5 shows the inhibitory effect of mangosteen extract on palm swelling in a carrageenen-induced paw edema model.

Fig. 6 shows the inhibitory effect of mangosteen extract on the affected part of arthritis in a collagen-induced arthritis animal model.

Claims (2)

Use of a compound of the following formula (I) for the manufacture of a medicament for regulating the secretion of cytokines, [wherein, both R ₁ and R ₂ are a methyl group] wherein the cytokine includes interleukins or tumor necrosis factor (TNF-α). The use according to claim 1, wherein the compound of formula (I) is isolated from Mangosteen ( Garcinia mangostana L.).